1. Field of the Invention
The present invention relates to a novel lipase, its production, and a method of hydrolyzing triglycerides by use of this novel lipase. The lipase of the present invention is used for hydrolyzing triglycerides and particularly, triglycerides present in serum. More particularly, the present invention relates to a method of hydrolyzing triglycerides by action of a lipase originating from the genus Penicillium on triglycerides contained in a biological body fluid sample such as serum. The method of the present invention is used for an assay of triglycerides present in a serum or the like.
2. Related Background Art
Methods have been known for an assay of triglycerides by hydrolyzing triglycerides present in a biological body fluid sample such as serum to determine a liberated fatty acid or glycerol, but almost all of the methods having been put into practical use are those for determining glycerol. Liberated glycerol is assayed after being further converted to detectable products by the action of glycerol kinase, glycerol dehydrogenase or glycerol oxidase.
The lipase first used for hydrolyzing triglycerides in serums was a lipase originating from pancreas of animals. However, this enzyme was not capable of perfectly hydrolyzing triglycerides. In order to perfectly hydrolyze triglycerides in serums, studies have been made searching for lipases derived from microorganisms, on the hydrolysis by a combination of lipases having different properties, and on the hydrolysis by a combination of a lipase with chemicals. For example, known are a lipase from Rhizopus arrhizus (Japanese Patent Publication No. 15638/1984 and Japanese Unexamined Patent Publication No. 25693/1977), a lipase from the genus Pseudomonas (Japanese Unexamined Patent Publications No. 50990/1974, No. 69186/1974, No. 89596/1974, No. 113695/1974, and No. 58898/1982), combined use of a lipase and a protease (Japanese Patent Publication No. 9518/1979 and Japanese Unexamined Patent Publication No. 114493/ 1978), a combination comprising a lipase from Rhizopus arrhizus, a carboxylesterase originating from pork liver, and an alkyl sulfate of an alkali metal or alkaline earth metal (Japanese Unexamined Patent Publication No. 64495/1974), a combination comprising a lipase from the genus Candida, a pancreas lipase, and a bile salt (Japanese Unexamined Patent Publication No. 11987/1977), a lipase from the genus Chromobacterium (Japanese Unexamined Patent Publications No. 68297/ 1976, No. 74692/1976 and No. 58898/1982), a combination comprising a lipase from Rhizopus arrhizus and a lipase from C. cylindracea (Japanese Unexamined Patent Publication No. 25694/1977 and Japanese Patent Publication No. 29/1981), a combination comprising a lipase from C. rugosa and a surface-active agent (Japanese Patent Publication No. 39158/1982), a combination comprising a lipase from Rhizopus arrhizus and a lipase from P. fluorescens (Japanese Patent Publication No. 28276/1982), a combination comprising a lipase from the genus Rhizopus or the like and a cholesterol esterase (Japanese Patent Publication No. 46799/1981), and a combination comprising a lipase and a surface-active agent, a phenol derivative or an aniline derivative (Japanese Patent Publication No. 5677/1983).
It is also known that a certain kind of lipases produced by the genus Pseudomonas yield about 2 moles of glycerol per 100 moles of fatty acid (Japanese Unexamined Patent Publication No. 187780/1984).
The present inventors do not know any report that a lipase originating from the genus Penicillium is effective for hydrolyzing serum triglycerides, but it is known that the genus Penicillium produces a lipase. Iwai et al report that a strain of Penicillium cyclopium Westring produces two kinds of lipases (Agr. Biol. Chem., Vol. 39, pp.1063-1070, 1975). They also report that a strain of Penicillium cyclopium M1 produces two kinds of lipases (J. Biochem., Vol. 87, pp. 205-211, 1980).
In the above prior art relating to the hydrolysis of triglycerides present in serum, the lipase originating from microorganisms when used alone, has been still unable to sufficiently hydrolyze triglycerides or has produced a slow rate of hydrolysis. In another aspect, there has been a drawback that the activities of these lipases may be inhibited by a surface-active agent contained in a reagent composition used for an assay of triglycerides. Also, the production thereof has been cumbersome when a plurality of lipases originating from different sources is used in combination.